Ace-inhibitor nitric salts

ABSTRACT

Compounds having platelet anti-aggregating activity and antihypertension activity having reduced branchial side effects.

This application is a 371 of PCT/EP98/03946 filed Jun. 24, 1998.

The present invention relates to products having an antihypertensiveactivity combined with a platelet-antiaggregating activity, andpharmaceutical compositions thereof.

In particular, it relates to products having an improvedantihypertensive activity and fewer side effecs, in particular in thebronchi, compared to the products currently being marketed asantihypertensive agents. The antihypertensive activity is combined witha platelet-antiaggregating activity.

Antihypertensive agents are known in the art. Particularly known are ACEinhibitors, which represent a first-choice pharmacological measure inthe treatment of cardiovascular diseases such as hypertension, angina,myocardial ischaemia, congestive heart failure, and others. ACEinhibitors act on the renin-angiotensin system which releasesangiotensin II, one of the most effective hypertensive agents known.More precisely, these drugs inhibit the activity of the angiotensinconverting enzyme, a carboxypeptidase which is mostly present in lungs,kidneys, and vessels. The action of this enzyme is not specific. Itinactivates plasma bradykinin, which possesses a vasodilatatoryactivity, and also helps diuresis and, in particular, natriuresis. Inother terms, plasma bradykinin possesses opposite effects compared tothose of angiotensin II. Therefore, ACE inhibitors prevent formation ofangiotensine II and, at the same time, degradation of bradykinin. Hence,ACE inhibitors certainly represent one of the most significantpharmacological innovation of the past few decades.

However, the administration of ACE inhibitors is often (about 20 to 30%of the cases) accompanied by side effects in the respiratory system,such as cough, dyspnea, broncho-constriction. Furthermore, these drugsshow a rather limited therapeutic profile, for example they have noplatelet-antiaggregating activity, so that, in the above cardiovasculartreatments, they are often associated with other drugs having anantiaggregating activity. For example, in the treatment of myocardialinfarction and prevention of relapses, it is essential to use a multiplecardiovascular therapy including, among others, the association of anantihypertensive with an antiaggregating agent.

It was felt the need for drugs with a better therapeutic profile andfewer side effects, in particular, at the respiratory system, forexample the bronchi.

The Applicant has unexpectedly and surprisingly found a specific classof ACE-inhibitor salts characterised by the fact that they possess,compared to other salts of the same compounds, a better antihypertensiveactivity and have fewer side effects in the bronchi.

An object of the present invention is, therefore, the nitric salts ofACE inhibitors having the following formulas:

in formula

or N—CH₃;

Y=CH₃, phenyl;

R^(III)=H,

R^(III) together with R^(IV) forms the following ring in the carbon atposition 4

R^(III) together with R^(V) (carbons at positions 4 and 5) forms thecyclohexane or cyclopentane rings

R^(IV)=H, or R^(IV) forms with R^(III) ring (IVa);

R^(V)=H, or a free valence, or R^(V) forms with R^(III) rings (IIIa) or(IIIb);

R^(VI)=H, or a single bond —O when R^(V) is a free valence so as to forma ketone group with the carbon atom at position 5.

The preferred nitrate salts of formula (I) include:

when X=C (R^(III)) (R^(IV)) as above defined, Y=phenyl,R^(III)=R^(IV)=R^(V)=R^(VI)=H, the residue of Enalapril;

as in Enalapril but with R^(III) which, together with R^(IV), forms ring(IVa), the residue of Spirapril;

as in Enalapril but with R^(III) which, together with R^(V), forms ring(IIIb), the residue of Ramipril;

as in Enalapril but with Y=CH₃ and R^(III) which, together with R^(V),forms ring (IIIa), the residue of Perindopril;

as in Enalapril, but with X=N—CH₃, R^(V) is a free valence and R^(VI)=—Oso as to form with carbon atom C₅ a ketone group, the residue ofImidapril.

The compounds of the classes of the invention, which are the precursorsof the salts, are used as optically-active single isomers or as mixturesthereof or in the form of racemates.

The precursor of class II is known as Lisinopril, that of class III isknown as Alacepril. The precursors are prepared according to the methodsdescribed in “The Mercx Index, Ed. 12”, herein incorporated byreference.

The salts of the present invention are prepared according to thefollowing method. The substance to be salified is dissolved in anorganic solvent, not containing in the molecule free hydroxyl groups,and then a stoichiometric amount of concentrated nitric acid is added.The salt is recovered by filtration and washed several times with asolvent, for example that used in the reaction. Polar organic solventsare preferred, such as , for example, acetonitrile, ethyl acetate, andothers.

It has surprisingly been found that the compounds of the presentinvention improve, compared to the same substances and ACE saltsgenerally, the pharmacological profile of the above ACE inhibitors and,additionally, exhibit a more favourable general and regionaltolerability.

The compounds of the present invention can be used as cardiovasculardrugs, in particular in the treatnent of hypertension, angina,myocardial ischaemia, congestive heart failure.

The salts of the present invention are formulated in the correspomdingpharmaceutical compositions according to the methods well known to thoseskilled in the art, which are, for example, described in Remington'sPharmaceutical Sciences, Ed. 15.

The examples below are meant to describe the invention and should not beunderstood as a limitation of same.

EXAMPLE 1 Synthesis of(S)-1-[N-[1-(ethoxycarbonyl)-3-phenylpropyl]-L-alanyl]-L-proline(Enalapril), and obtainement of the nitrate salt in acetonitrile

A mixture of ethyl-2-oxo-4-phenylbutyrate (2.1 g) and L-alanyl-L-proline(0.4 g) in ethanol/water 1/1 was treated slowly at room temperature witha solution of sodium cyanoborohydride (0.4 g) in ethanol/water 1/1.

At the end of the reaction, the product was absorbed on a strong acidion exchange resin and eluted with an aqueous solution containing 2%(v/v) of pyridine. The fractions which contained the product werelyophilised to obtain the crude compound. Chromatography then allowedisolation of the desired isomer (−) practically pure (0.24 g).

The isomer was then dissolved in acetonitrile and treated, maintainingthe reactor in an ice bath, with a stoichiometric amount of concentratednitric acid dissolved in acetonitrile. After cooling and filtration, thesolid was washed with cold acetonitrile and a 97%-pure (HPLC: highpressure liquid chromatography) Enalapril nitric salt was recovered. A99%-pure (HPLC) salt could be obtained by crystallisation fromacetonitrile.

EXAMPLE 2 Synthesis of Enalapril, and Obtainement of the Nitrate Salt inEthyl Acetate

A mixture of ethyl-2-oxo-4-phenylbutyrate (15 g), L-alanyl-L-proline (9g), molecular sieves 3 A° (40 g) and Raney nickel (10.8 g) in ethanol(300 ml) was hydrogenated at room temperature and at a pressure of about3 atm. up to the hydrogen is not-consumed any more. After filtration ofthe undissolved substance (washing well with ethanol, the solvent wasevaporated under vacuunm to obtain a mixture of diastereoisomers formedof 85% by the expected product (by HPLC). The obtained product wasdissolved in a mixture made up of 200 ml of water and 70 ml of methylacetate. By keeping the solution under stirring, the pH was adjusted to8.6 with 50% NaOH. The organic phase was separated and the aqueous phasewas thoroughly washed with ethyl acetate (3×50 ml). The aqueous phasewas adjusted to pH 4.3 with hydrochloric acid, saturated with sodiumchloride and then extracted with ethyl acetate (4×100 ml). After dryingwith sodium sulphate and evaporating the solvent off under vacuum, theresidue was dissolved in ethyl acetate maintaining the reactor in an icebath, and salified by treating with a stoichiometric amount ofconcentrated nitric acid. After stirring for two hours, it was cooled,filtered, washed with ethyl acetate and recrystallised from acetonitrileto obtain 12.5 g of nitric salt of the isomer (−), about 99%-pure (byHPLC).

EXAMPLE 3 Acute Toxicity

A group of 10 mice (weight 15 to 25 g) received a single oral dose of100 mg/Kg. All the animals survived during the observation period (14days). No toxicity symptom was observed.

EXAMPLE 4 Antihypertensive Activity

The antihypertensive activity of the nitrate salts of the compounds ofthe invention was determined in accordance with the method of Laubie etal., J. Cardiovasc. Pharmacol. 6, 1076, 1984. No 6 rats weighing about200 to 250 g were used per experimental group. Four groups were formed,which were intraperitoneally treated respectively as shown below:

Enalapril maleate 100 μg/Kg Enalapril maleate 300 μg/Kg Enalaprilnitrate 100 μg/kg Enalapril nitrate 300 μg/kg

The doses are referred to the amount of Enalapril (cation) in the salt.The antihypertensive response was evaluated as per-cent inhibition ofthe hypertension induced by the administration of a dose of 100 μg/Kgi.v. of angiotensin I as described in the above article.

The results are shown in Table I

TABLE I Inhibition % for DOSE angiotensin-I- COMPOUND (μg/Kg/i.p.)induced hypertension Enalapril maleate 100 18 Enalapril maleate 300 55Enalapril nitrate 100 35 Enalapril nitrate 300 67

EXAMPLE 5 Pharmacological Effects of the Salts of the Invention onBronchial Spasm Induced by Administration of Substance P

Activity was evaluated measuring the strengthening of bronchial spasminduced by substance P, determined in accordance with the method ofSubissi et al., Br. J. Pharmacol. 100, 502-6, 1990. The model describedby Subissi is predictive of bronchial side effects due to theadministration of ACE inhibitors.

Four groups (6 animals/group) of female Guinea pigs weighing about 300to 400 g were anaesthetised with ethyl urethane (200 mg/Kg) underartificial pressure at constant positive pressure. The compounds wereadministered intra-peritoneally 30 minutes before substance P. The saltdoses administered were the same as in Example 4. The changes in tidalair were then measured in accordance wuth the method of Konzett, Arch.Exp. Pathol. Pharmacol. 195, 71, 1940, before and after theadministration of substance P (200 μg/Kg), with or without the testsalts, i.e. Enalapril maleate and nitrate.

The results are shown in Table II

As seen from the data, Enalapril nitrate possessed a better respiratoryprofile than Enalapril maleate at both tested doses.

TABLE II Tidal air change % in DOSE bronchial spasm induced by COMPOUND(μg/Kg/i.p.) substance P Enalapril maleate 100 +16 Enalapril maleate 300+28 Enalapril nitrate 100  −5 Enalapril nitrate 300  −7

EXAMPLE 6 Platelet-antiaggregating Activity

The in-vivo model described by Pinon et al., J. Pharm. Methods 12,79-84, 1984, was used.

Two groups of 6 rats each, weighing about 200 to 250 g, were treatedwith an oral. dose of 10 mg/Kg/die of Enalapril maleate or nitraterespectively (the dose is referred to the amount of Enalapril cation inthe salt) for five days, while a third group acted as a control group.About 18 hours before the last treatment, the animals were fasted. Onehour after this treatment the animals were anaesthetised with 10% ethylurethane (1 g/Kg intraperitoneally) and the left jugular vein and theright carotid artery were cannulated. Collagen (type 6, Sigma) was thenadministered intravenously at a dose of 2 mg/Kg. Three minutes later twoblood samples, A and B, were collected from the carotid artery of eachanimal.

1.6 ml of EDTA/formalin buffer (24 mM tetrasodium EDTA, 1.3 mM KH₂PO₄,13.4 mM Na₂HPO₄) was added to the first sample (sample A) containing 0.4ml of blood.

The second blood sample (sample B) had the same volume as the previoussample (0.4 ml of blood) but, instead of the buffer, 1.6 ml of a salinesolution (physiological NaCl solution) was added.

The samples were then transferred into 5-ml test tubes and allowed tostand at room temperature for 15 minutes.

A microscope platelet count was then performed. The platelet count insamples B and A represent the total number of platelets and the totalnumber of aggregated platelets respectively. The results shown in TableIII are expressed as a % of platelet aggregation and are referred to the% value obtained in the control group.

TABLE III DOSE/die Antiaggregating COMPOUND (mg/Kg/os) activity %Enalapril maleate 10  5 Enalapril nitrate 10 58

What is claimed is:
 1. Nitric salts of ACE inhibitors having thefollowing formulas:

in formula

or N—CH₃ Y=CH₃, or phenyl; R^(III)=H, R^(III) together with R^(IV) formsthe following ring in the carbon at position 4

R^(III) together with R^(V) (carbons at positions 4 and 5) forms thecyclohexane or cyclopentane rings

R^(IV)=H, or R^(IV) forms with R^(III) ring (IVa); R^(V)=H, or a freevalence or R^(V) forms with R^(III) rings (IIIa) or (IIIb); R^(VI)=H, ora single bond —O when R^(V) is a free valence so as to form a ketonegroup with the carbon atom at position
 5. 2. Nitric salts according toclaim 1, wherein, in formula (I), X=C (R^(III)) (R^(IV)), Y=phenyl,R^(III)=R^(IV)=R^(V)=R^(VI)=H, the residue of Enalapril; as in Enalaprilbut with R^(III) which, together with R^(IV), forms ring (IVa), theresidue of Spirapril; as in Enalapril but with R^(III) which, togetherwith R^(V), forms ring (IIIb), the residue of Ramipril; as in Enalaprilbut with Y=CH₃ and R^(III) which, together with R^(V), forms ring(IIIa), the residue of Perindopril; as in Enalapril, but with X=N—CH₃,R^(V) is a free valence and R^(VI)=—O so as to form with carbon atom C₅a ketone group, the residue of Imidapril.
 3. Nitric salts according toclaim 2, wherein, in formula (I), X=C (R^(III)) (R^(IV)), Y=phenyl,R^(III)=R^(IV)=R^(V)=R^(VI)=H, the residue of Enalapril.
 4. Apharmaceutical composition containing an effective amount of one or morenitrate salts of compounds I, II or III of claim 1 and apharmaceutically acceptable excipient or carrier.
 5. A method oftreating hypertension in an individual comprising administering atherapeutically effective amount of compounds I, II or III according toclaim
 1. 6. A method for treating a patient in need of a plateletantiaggregating treatment comprising administering to said patient aneffective amount of compounds I, II or III according to claim
 1. 7. Amethod for treating angina in an individual comprising administering atherapeutically effective amount of compounds I, II or III according toclaim
 1. 8. A method for treating myocardial ischaemia in an individualcomprising administering a therapeutically effective amount of compoundsI, II or III according to claim
 1. 9. A method for treating congestiveheart failure in an individual comprising administering atherapeutically effective amount of compounds I, II or III according toclaim 1.